RESUMO
Cystic fibrosis (CF) is one of the most common recessive conditions among whites, with an estimated carrier frequency of 1 in 25 in the United States. Population-based CF carrier screening was implemented in the United States in 2001. The number of mutations screened by each laboratory may vary; however, the 23 most common CF mutations recommended for screening by the American College of Medical Genetics and American College of Obstetricians and Gynecologists are included in all platforms. The c.1364C>A (p.A455E) mutation located in exon 10 of the CFTR gene is one of the 23 mutations. Because CFTR exon 10 and its flanking intronic regions are duplicated and transposed onto several other chromosomes of the human genome during evolution and function as unprocessed pseudogenes, variations in the CFTR pseudogenes may confound CF screening results for mutations located in exon 10 of the CFTR gene. We report an incorrectly identified carrier status for the c.1364C>A (p.A455E) mutation in a healthy individual using the Hologic InPlex CF assay. Further analysis revealed that the mutation resides in one of the CFTR pseudogenes. Because most commercial kits and laboratory-developed tests for CF carrier screening involve a short amplicon encompassing this mutation, this finding suggests that individuals with the c.1364C>A (p.A455E) mutation may require further investigation to avoid a false assignment of CF carrier status.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/diagnóstico , Fibrose Cística/genética , Mutação/genética , Pseudogenes/genética , Adulto , Sequência de Bases , Análise Mutacional de DNA/métodos , Testes Genéticos/métodos , Heterozigoto , Humanos , Masculino , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Estados UnidosRESUMO
The TWEAK receptor Fn14 (TNFRSF12), a member of the TNF Receptor superfamily, can mediate many processes, including apoptosis. Fn14 agonists have therefore been the subject of interest as potential cancer therapeutics. In cell culture experiments, interferon gamma (IFNγ) is typically required for induction of apoptotic activity by either TWEAK or Fn14 agonistic antibodies in most cell lines. We have investigated the mechanism of IFNγ signaling and the role of JAK-STAT signaling in TWEAK/Fn14-mediated tumor cell killing. We found that IFNγ-mediated enhancement of tumor cell killing is JAK-STAT dependent, as JAK inhibitors block IFNγ-dependent TWEAK induced apoptosis. Exposure of tumor cells to IFNγ results in an increase in Fn14 expression on the cell surface, which may be a mechanism by which IFNγ induces sensitivity to TWEAK. In a reciprocal fashion, we observed that IFNγ receptor levels increase in response to TWEAK treatment in WiDr cells. Significantly, we found that TWEAK alone can induce STAT1 phosphorylation in WiDr tumor cells. Moreover, TWEAK induction of tumor cell apoptosis in WiDr cells in the absence of IFNγ is mediated by the JAK-STAT pathway. Correspondingly, we show that treatment of tumor bearing mice with mBIIB036, an Fn14 agonistic antibody, results in STAT1 phosphorylation in the tumors. Notably, the level of STAT1 phosphorylation appears to correlate with the degree of tumor growth inhibition by BIIB036 in vivo. Additionally, in WiDr cells, TWEAK induces a soluble factor, which we have identified as IFNß, capable of independently inducing STAT1 phosphorylation when transferred to naïve cells. Finally, either IFNα or IFNß can partially substitute for IFNγ in sensitizing tumor cells to Fn14 agonists. In summary, we show that TWEAK/Fn14 can signal through the JAK-STAT pathway to induce IFNß, and that the ability of TWEAK to induce tumor cell apoptosis is mediated by JAK-STAT signaling. We also demonstrate that IFNγ enhancement of TWEAK/FN14-mediated tumor cell death is JAK-dependent and may occur by IFNγ-dependent upregulation of Fn14 on tumor cells. These findings may have implications for the appropriately targeted clinical development of Fn14 agonists as anti-cancer therapy.
Assuntos
Interferon gama/metabolismo , Janus Quinases/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Transcrição STAT1/metabolismo , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Interferon gama/farmacologia , Janus Quinases/antagonistas & inibidores , Camundongos , Fosforilação , Ligação Proteica , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/metabolismo , Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Receptores do Fator de Necrose Tumoral/imunologia , Transdução de Sinais , Receptor de TWEAKRESUMO
INTRODUCTION: the development of new drugs over the last few decades has targeted specific proteins thought to be a key to the disease state. MAPK kinases 1 and 2 (commonly known as MEK1-2) represent such proteins as they lie downstream of important drug targets for oncology, such as EGFR, RAS and RAF. Several MEK1-2 inhibitors are currently in Phase I and II clinical trials in oncology. AREAS COVERED: this review of current literature and recent conferences provides a background on the RAS-RAF-MEK-ERK signaling pathway and a discussion of early MEK inhibitors. The potential of MEK1-2 inhibitors for the treatment of inflammation is briefly presented. Preclinical and early clinical results are discussed for MEK inhibitors currently in development. Completed clinical trials of MEK inhibitors in oncology include some disappointments as well as some promising signs of the value of these compounds and we discuss the potential for MEK inhibitors as monotherapy and their use in drug combinations. EXPERT OPINION: the utility of MEK inhibitors as anticancer agents will depend on careful patient selection based on the presence of mutations in genes such as KRAS and BRAF, the identification of additional predictive biomarkers, and an improved understanding of the benefit of drug combinations utilizing both established and emerging therapeutics.
Assuntos
Antineoplásicos/uso terapêutico , Inflamação/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Ensaios Clínicos como Assunto , Genes ras , Humanos , Sistema de Sinalização das MAP Quinases/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Transdução de Sinais , Quinases raf/antagonistas & inibidores , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ras/antagonistas & inibidores , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
BACKGROUND: Combining MEK inhibitors with other signalling pathway inhibitors or conventional cytotoxic drugs represents a promising new strategy against cancer. RDEA119/BAY 869766 is a highly potent and selective MEK1/2 inhibitor undergoing phase I human clinical trials. The effects of RDEA119/BAY 869766 as a single agent and in combination with rapamycin were studied in 3 early passage primary pancreatic cancer xenografts, OCIP19, 21, and 23, grown orthotopically. METHODS: Anti-cancer effects were determined in separate groups following chronic drug exposure. Effects on cell cycle and downstream signalling were examined by flow cytometry and western blot, respectively. Plasma RDEA119 concentrations were measured to monitor the drug accumulation in vivo. RESULTS: RDEA119/BAY 869766 alone or in combination with rapamycin showed significant growth inhibition in all the 3 models, with a significant decrease in the percentage of cells in S-phase, accompanied by a large decrease in bromodeoxyuridine labelling and cell cycle arrest predominantly in G1. The S6 ribosomal protein was inhibited to a greater extent with combination treatment in all the three models. Blood plasma pharmacokinetic analyses indicated that RDEA119 levels achieved in vivo are similar to those that produce target inhibition and cell cycle arrest in vitro. CONCLUSIONS: Agents targeting the ERK and mTOR pathway have anticancer activity in primary xenografts, and these results support testing this combination in pancreatic cancer patients.
Assuntos
Difenilamina/análogos & derivados , MAP Quinase Quinase Quinases/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Sirolimo/administração & dosagem , Sulfonamidas/administração & dosagem , Animais , Antineoplásicos/farmacologia , Bromodesoxiuridina/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Difenilamina/administração & dosagem , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Transdução de SinaisRESUMO
A number of conditions, including osteoporosis, frailty, and sexual dysfunction in both men and women have been improved using androgens. However, androgens are not widely used for these indications because of the side effects associated with these drugs. We describe an androgen receptor (AR) ligand that maintains expected anabolic activities with substantially diminished activity in the prostate. LGD2226 is a nonsteroidal, nonaromatizable, highly selective ligand for the AR, exhibiting virtually no affinity for the other intracellular receptors. We determined that AR bound to LGD2226 exhibits a unique pattern of protein-protein interactions compared with testosterone, fluoxymesterone (an orally available steroidal androgen), and other steroids, suggesting that LGD2226 alters the conformation of the ligand-binding domain. We demonstrated that LGD2226 is fully active in cell-based models of bone and muscle. LGD2226 exhibited anabolic activity on muscle and bone with reduced impact on prostate growth in rodent models. Biomechanical testing of bones from animals treated with LGD2226 showed strong enhancement of bone strength above sham levels. LGD2226 was also efficacious in a sex-behavior model in male rats measuring mounts, intromissions, ejaculations, and copulation efficiency. These results with an orally available, nonaromatizable androgen demonstrate the important role of the AR and androgens in mediating a number of beneficial effects in bone, muscle, and sexual function independent from the conversion of androgens into estrogenic ligands. Taken together, these results suggest that orally active, nonsteroidal selective androgen receptor modulators may be useful therapeutics for enhancing muscle, bone, and sexual function.
Assuntos
Aminoquinolinas/farmacologia , Copulação/efeitos dos fármacos , Vértebras Lombares/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Próstata/efeitos dos fármacos , Quinolonas/farmacologia , Administração Oral , Aminoquinolinas/síntese química , Aminoquinolinas/metabolismo , Animais , Antineoplásicos Hormonais/farmacologia , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Fluoximesterona/farmacologia , Humanos , Vértebras Lombares/fisiologia , Masculino , Orquiectomia , Osteossarcoma , Neoplasias da Próstata , Quinolonas/síntese química , Quinolonas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Androgênicos/metabolismo , SpodopteraRESUMO
Nuclear receptors (NRs) are ligand-regulated transcription factors important in human physiology and disease. In certain NRs, including the androgen receptor (AR), ligand binding to the carboxy-terminal domain (LBD) regulates transcriptional activation functions in the LBD and amino-terminal domain (NTD). The basis for NTD-LBD communication is unknown but may involve NTD-LBD interactions either within a single receptor or between different members of an AR dimer. Here, measurement of FRET between fluorophores attached to the NTD and LBD of the AR established that agonist binding initiated an intramolecular NTD-LBD interaction in the nucleus and cytoplasm. This intramolecular folding was followed by AR self-association, which occurred preferentially in the nucleus. Rapid, ligand-induced intramolecular folding and delayed association also were observed for estrogen receptor-alpha but not for peroxisome proliferator activated receptor-gamma2. An antagonist ligand, hydroxyflutamide, blocked the NTD-LBD association within AR. NTD-LBD association also closely correlated with the transcriptional activation by heterologous ligands of AR mutants isolated from hormone-refractory prostate tumors. Intramolecular folding, but not AR-AR affinity, was disrupted by mutation of an alpha-helical ((23)FQNLF(27)) motif in the AR NTD previously described to interact with the AR LBD in vitro. This work establishes an intramolecular NTD-LBD conformational change as an initial component of ligand-regulated NR function.